Metabolic syndrome (MetS) is a constellation of metabolic disorders that increase the incidence of cardiovascular diseases. Risks of macrovascular complications are significantly enhanced in individuals with MetS. However, the molecular basis for accelerated atherosclerosis in MetS remains poorly understood. Hyperglycemia and hyperleptinemia, co-existing features of MetS, are independent risk factors for development of atherosclerotic vascular disorders. Thrombospondin-1 (TSP-1) is a matricellular protein with increased expression in diabetes, obesity and MetS. Multiple clinical studies, including animal data, highlight a role of TSP-1 in vascular pathology. Our data suggest that a TSP-1-dependent mechanism, specific to MetS where hyperglycemia and hyperleptinemia co-exist, mediates accelerated vasculopathy in MetS. However, the precise mechanism by which TSP-1 drives vascular disease in MetS is unclear. Relevant to this application are observations that in a murine model of combined MetS and atherosclerosis, TSP-1 expression correlates with atherosclerotic lesion formation and increased smooth muscle cell (SMC) de-differentiation to a synthetic proliferative phenotype. Accordingly, the overarching goal of this proposal is to delineate the regulatory mechanism of TSP-1 on SMC phenotypic plasticity and lesion pathogenesis in MetS. We hypothesize that TSP-1 induces VSMC de-differentiation to an atherogenic phenotype via activation of specific transcriptional pathways in MetS. We further postulate that targeted TSP-1 deletion will blunt these pathways blocking atherosclerotic complications in MetS. The specific aims are: 1) We will determine the molecular mechanism by which TSP-1 regulates VSMC phenotypic switching in metabolic syndrome, 2) We will interrogate whether TSP-1 regulates lesion pathogenesis in metabolic syndrome. This will be tested via loss of function approaches using mouse models of MetS and combined MetS with atherosclerosis, with genetic TSP-1 deletion, as well as aortic SMC primary cultures in vitro derived from MetS mice. In addition, we will perform SMC lineage tracing studies in vivo using the atherosclerotic mouse model of MetS with TSP-1 deletion, expressing Cre-inducible SMC-specific reporter gene (ROSA26-eYFP), with SMCs genetically labeled with yellow fluorescent protein (YFP). We will utilize different biochemical, histological and molecular biology approaches in combination with High-frequency Ultrasound Imaging, en-face atherosclerotic lesion assay, HDL function studies as well as metabolic phenotyping using EchoMRI and CLAMS. Overall, this proposal will advance our fundamental understanding of the molecular basis of atherosclerotic vascular disease in MetS and open novel avenues for development of TSP-1-targeted therapies for the treatment of MetS-induced vasculopathy. Of relevance to R15 AREA grants, this project will provide an excellent platform to advance undergraduate and graduate student research, strengthening the research setting of our institute.